Sequence dependent conformations of oligomeric DNA's in aqueous solutions and in crystals

In order to determine the sequence dependence of the conformation of deoxynucleotides, Raman spectra have been obtained for the following oligodeoxynucleotides in aqueous salt solutions and in crystals: d(CpG)(I), d(TGCGCGCA)(II), d(CACGCGTG)(III), d(CGTGCACG)(IV), d(CGCATGCG)(V), d(ACGCGCGT)(VI), d(CGCGTACGCG)(VII), d(CGCACGTGCG)(VIII) and d(CGTGCGCACG)(IX), d(GCTATAGC) (X), d(GCATATGC) (XI), d(GGTATACC) (XII) and d(GGATATCC) (XIII). The normal B type conformation is observed for all the oligomer DNA's at low salt (0.1-1.0 M NaCl) concentration in the temperature range of 0-25 degrees C. It was considered possible that all of the first nine oligomers could go into the Z form in aqueous high salt (5.0-6.0 M NaCl) solutions, and under these conditions the last four were considered candidates to go into the A form. The B-type conformation was found to exist in high salt solutions for (I), (IV), (V), (VI), (X), (XI) and (XIII); the Z or partial Z conformation appears in high salt solution for the oligomers, (II), (III), (VII), (VIII) and (IX); an A or partial A conformation appears in high salt solution for (XII). In the crystalline state, (IV), (VIII), (X), and (XI) stay in the B-form and all of the other oligomers adopt the complete Z-form except for (XII) which crystallizes in the A form. In both the crystal and in aqueous solutions, the identification of the conformation genus was made by means of Raman spectroscopy. In the crystal of (I), grown at pH7.0, guanosine is found to be in C3'-endo/syn conformation and cytidine in C2'-endo/anti, which may be taken as the ideal building block of the typical Z conformation. At pH4, (I) crystallizes in a conformation similar to the B genus. A study of the thermally induced B to Z transition has been carried out for (II) and (III). Based on the analysis of Raman spectra of the alternating pyrimidine-purine oligomers which might be expected to go into the Z form, the tendency for these oligonucleotides to adopt the Z form can be ranked as: d(CGCGCGCG) greater than (II) greater than (III) greater than (V) approximately (VI) greater than (IV) for octamers and (VII) greater than (VIII) greater than (IX) for the decamers. Similarly, those oligomers which might have a tendency to go into the A form could be ranked as (XII) greater than (XIII) approximately (X) greater than (XI). These data should provide help in formulating rules for predicting the sequence dependence of the B to A and B to Z transitions. Some possible rules are explored, but precautions should be taken.     

The high salt form of poly(dG-dC).poly(dG-dC) is left-handed Z-DNA: Raman spectra of crystals and solutions.

The laser-Raman spectra of crystalline d(CpGpCpGpCpG) and of aqueous poly(dG-dC).poly(dG-dC) in high salt (4M NaCl) and low salt (0.1M NaCl) solutions have been measured and compared. The spectra of the crystal and the high-salt solution show a striking congruence, which indicates clearly that the high-salt form of the aqueous polymer has the left-handed Z-DNA structure of the crystalline oligomer. These two spectra differ substantially from that of the low-salt form of the polymer, which has been found previously to have spectral characteristics of the B-form of DNA. The high salt spectrum shows a unique line due to guanine residues at 625 cm-1 which should be useful for qualitative and possibly quantitative assessment of the amount of Z-structure present in a sample of DNA.     

Characterization of DNA structures by Raman spectroscopy: high-salt and low-salt forms of double helical poly(dG-dC) in H2O and D2O solutions and application to B, Z and A-DNA.

Raman spectra of poly(dG-dC) . poly(dG-dC) in D2O solutions of high (4.0M NaCl) and low-salt (0.1M NaCl) exhibit differences due to different nucleotide conformations and secondary structures of Z and B-DNA. Characteristic carbonyl modes in the 1600-1700 cm-1 region also reflect differences in base pair hydrogen bonding of the respective GC complexes. Comparison with A-DNA confirms the uniqueness of C = O stretching frequencies in each of the three DNA secondary structures. Most useful for qualitative identification of B, Z and A-DNA structures are the intense Raman lines of the phosphodiester backbone in the 750-850 cm-1 region. A conformation-sensitive guanine mode, which yields Raman lines near 682, 668, or 625 cm-1 in B (C2'-endo, anti), A (C3'-endo, anti) or Z (C3'-endo, syn) structures, respectively, is the most useful for quantitative analysis. In D2O, the guanine line of Z-DNA is shifted to 615 cm-1, permitting its detection even in the presence of proteins.     

Conformational Transitions of Poly(dl-dC) in Aqueous Solution as Studied by Ultraviolet Resonance Raman Spectroscopy

Abstract

Poly(dI-dC) in aqueous solution can undergo different equilibrium geometries, which strongly depend on salt nature and concentrations. These equilibrium structures have been monitored by resonance Raman spectroscopy (RRS) measurements in the ultraviolet region, i. e. by using 257 and 281 nm laser excitation wavelengths which favor the resonance enhancement of the Raman contributions from inosine and cytosine residues of poly(dI-dC), respectively. Spectral changes depending on the NaCl concentration and on the presence of Ni²⁺ ions have been observed and interpreted in comparison with RRS results previously obtained for other alternating purine-pyrimidine polydeoxyribonucleotides, i.e. poly(dG-dC), poly(dA- dT) and poly(dA-dC). poly(dG-dT), which also showed B to Z conformational transitions in varying the salt concentrations. It is shown here that: i) the base stacking geometries are nearly the same in the high-salt form (5 M NaCl) of poly(dl-dC) as in the low-salt form (0.1M NaCl) of the polymer, ii) however, the high-salt structure yields important differences from a B-helix (obtained in low-salt solution) as regards the nucleoside conformations (sugar puckering and base-sugar orientation), and: iii) the addition of 9 mM NiCl₂ in the high-salt (5 M NaCl) solution of poly(dI-dC) induces the Z-conformation of the polymer.     

Z form of poly d(A-T).poly d(A-T) in solution studied by CD and UV spectroscopies

Near UV CD spectra, UV absorption spectra and their first derivatives have been recorded on poly d(A-T).poly d(A-T) solutions in presence of high NaCl concentration and various amounts of NiCl2. Comparison of the results presented here with those obtained for poly d(G-C).poly d(G-C) and poly d(A-C).poly d(G-T) in comparable conditions, and the I.R. and Raman data on poly d(A-T).poly d(A-T), allows us to assign the new spectra to the Z conformation of poly d(A-T).poly d(A-T) in solution. The mechanism by which nickel ions induce the B----Z interconversion in the presence of high NaCl concentration is discussed.     

Secondary structure of calsequestrin in solutions and in crystals as determined by Raman spectroscopy

Calsequestrin has been precipitated with calcium into five different crystal forms: cruciform twins, flat rectangles, thin needles, bipyramids, rectangular prisms, and a sixth precrystalline form, spheres. Raman spectra of the spheres and the cruciform twins are the same. The Raman spectrum of a physiological concentration (10%) of calsequestrin in calcium-free solution is the same as the spectrum of calcium precipitated calsequestrin in the amide I region, and in the C-C stretching region, but these spectra are different in the amide III region. The Raman spectrum of unfolded calsequestrin in 5 M guanidine hydrochloride is quite different from the other spectra, but it is not similar to the spectra of other unfolded proteins. Estimates of secondary structure from the amide I region indicate that calsequestrin in calcium-free solution and calcium-precipitated forms has 40 +/- 5% helix, 30 +/- 4% beta-strand, and 18 +/- 2% reverse turn. Secondary structure estimates calculated from the amide III region are not significantly different. They indicate 41 +/- 5% helix and 36 +/- 6% beta-strand for the precipitated forms, and 32 +/- 5% helix and 39 +/- 6% beta-strand for solutions. Calsequestrin unfolded in 5 M guanidine hydrochloride at 100 mg/ml gives 24 +/- 5% helix and 48 +/- 6% beta-strand.     

No pathophysiologic relationship of soluble biliary proteins to cholesterol crystallization in human bile

This study explores the pathophysiologic effects of soluble biliary glycoproteins in comparison to mucin gel and cholesterol content on microscopic crystal and liquid crystal detection times as well as crystallization sequences in lithogenic human biles incubated at 37 degrees C. Gallbladder biles from 13 cholesterol gallstone patients were ultracentrifuged and microfiltered (samples I). Total biliary lipids were extracted from portions of samples I, and reconstituted with 0.15 m NaCl (pH 7.0) (samples II). Portions of samples II were supplemented with purified concanavalin A-binding biliary glycoproteins (final concentration = 1 mg/mL) (samples III), or mucin gel (samples IV), respectively, isolated from the same cholesterol gallstone biles. Samples V consisted of extracted biliary lipids from uncentrifuged and unfiltered bile samples reconstituted with 0.15 m NaCl (pH 7.0). Analytic lipid compositions of samples I through IV were identical for individual biles but, as anticipated, samples V displayed significantly higher cholesterol saturation indexes. Detection times of cholesterol crystals and liquid crystals were accelerated in the rank order of samples: IV > V > I = II = III, indicating that total soluble biliary glycoproteins in pathophysiologic concentration had no appreciable effect. Crystallization sequences (D. Q-H. Wang and M. C. Carey. J. Lipid Res. 1996. 37: 606-630; and 2539-2549) were similar among samples I through V. Crystal detection times and numbers of solid cholesterol crystals were accelerated in proportion to added mucin gel and the cholesterol saturation of bile only.For pathophysiologically relevant conditions, our results clarify that mucin gel and cholesterol content, but not soluble biliary glycoproteins, promote cholesterol crystallization in human gallbladder bile.     

Recognition of a guanine-cytosine base pair by 8-oxoadenine.

Two oligodeoxyribonucleotides, d-CTTCTTTTTTATTTT, I(A), and d-ATTATTTTTTATTTT, II(A), where C is 5-methylcytosine and A is 8-oxoadenine, were prepared and their interactions with the duplex d-GAAGAAAAAAYAAAA/d-TTTTZTTTTTTCTTC, III.IV(Y.Z), were studied. Oligomers I(A) and II(A) each form triplexes with III.IV(G.C) at temperatures below 20 degrees C as shown by continuous variation experiments, melting experiments, and circular dichroism (CD) spectroscopy. The CD spectra of these triplexes are almost identical to those formed by I(C) and II(C), oligomers which contain cytosine in place of 8-oxoadenine. This suggests that the 8-oxoadenine-containing triplexes have conformations which are very similar to those of the cytosine-containing triplexes. The melting temperature (Tm) for dissociation of the third strand of triplex II.III.IV(A.G.C) is 22 degrees C at pH 7.0 and 8.0, whereas the Tm of the corresponding transition in triplex II.III.IV(C.G.C) decreases from 28 degrees C at pH 7.0 to 17 degrees C at pH 8.0. The pH dependence of the Tm in the latter triplex reflects the necessity of protonating the N-3 of cytosine in order for it to form two hydrogen bonds with G of the G.C base pair. It appears that the keto form of 8-oxoadenine can potentially form two hydrogen bonds with the N-7 and O-6 atoms of G of the G.C base pair, when the 8-oxoadenine is in the syn conformation and in contrast to cytosine does not require protonation of the base. Oligomer I(A) does not form triplexes with III.IV(Y.Z) when Y.Z is A.T or T.A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of conformation characteristics of polydeoxyribonucleotides with non-random base sequence by slow 1H----3H exchange

The rate constants of 1H----3H exchange between water and C8H-groups of purinic residues of alternating polynucleotides: poly[d(A-T)].poly[d(A-T)] (I), poly[d(G-C)].poly[d(G-C)] (II), poly[d(A-C)].poly[d(G-T)] (III) and homopolynucleotides: poly(dA).poly(dt) (IV), poly(dG).poly(dC) (V), as well as DNA E. coli, was determined in 0.15 M NaCl at 25 degrees C. The retardation of exchange observed at these conditions (compared to that of the B-form DNA) is in agreement with the model of B-alternating structure for the (I) and is attributed to the co-existence of B- and A-conformers for the (V) in solution. Absence of distinguishable differences in exchange rate constants for purinic residues of the (II), (III) and (IV) (compared to that of the B-form DNA) evidences that conformations of these polynucleotides in solution are similar to "canonical" B-form DNA and don't correlate with the model of "heteronomous" DNA which was proposed for (IV).     

Characterization of calcium-independent forms of protein kinase C-beta in phorbol ester-treated rabbit platelets

The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms.     

The length of a junction between the B and Z conformations in DNA is three base pairs or less

Recently it has been suggested that double-helical complexes formed between the DNA sequences (CG)n(A)m and their conjugates, (T)m(CG)n, would be candidates for the formation of a B-Z junction in aqueous solution at high salt concentrations [Peticolas et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 2579-2583]. The junction was predicted to occur between a B-type helix in the d(A)m.d(T)m section and a Z-type helix in the self-complementary (CG)n.(CG)n sequence. In this paper we report Raman experiments on the deoxyoligonucleotides d(CGCGCGCGCGCGAAAAA) and d(CGCGCGAAAAA) and their complements. It is found the latter compound cannot be induced into the Z form in saturated salt solution but that the former sequence goes into a B-Z junction at 5.5 M salt. From a comparison of the relative intensity of the Raman conformational marker bands for B and Z DNA for both the A-T and C-G base pairs, it is shown that in 5.5 M NaCl solution none of the A-T base pairs are in the Z form, but nine of the C-G base pairs are in the Z form. The remaining three C-G base pairs are either in the junction or in the B form. Thus, the junction is formed from three or less C-G base pairs. If the solution is made 95 microM with NiCl2, then the entire duplex goes into the Z form and the Raman bands of the adenine are completely changed into those of the Z form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Z helix-coil transition of d(C-Br8G-C-G-C-Br8G) studied by CD, 1H-NMR and IR spectroscopies

The conformation of d(C-Br8G-C-G-C-Br8G) in aqueous solution was studied by CD and 1H-NMR spectroscopy and in condensed phase by IR spectroscopy. Whether in 0.1 M or 3 M NaCl solution or in film the only double helical structure adopted by brominated d(C-G)3 oligomer is the Z form. The IR spectrum of the film presents all the characteristic absorptions of the Z conformation and in particular is indicative of a syn conformation for the central guanosine as well as for the brominated one. Imino proton resonances of d(C-Br8G-C-G-C-Br8G) demonstrating the duplex formation were observed up to 60 degrees C. It is interesting to note that the significant highfield shifts of the dC H5" exocyclic sugar protons characteristic of the non exchangeable proton spectra of d(C-G)3 containing 5-methyl dC residues in the Z form were also detected in the proton spectrum of brominated oligomer. Whereas formation of the Z helix of methylated d(C-G)3 oligomers dependent on the salt concentration was found to occur via the preliminary formation of a B helix even in 4 M NaCl solution, the Z helix of d(C-Br8G-C-G-C-Br8G) is obtained directly from the coil form. However, IR data suggest that in the Z form of d(C-Br8G-C-G-C-Br8G), the overlapping of the base planes should be slightly different in comparison with the stacking observed in d(C-G)3 crystals. The kinetic data (activation energy and lifetime) of the Z helix-coil transition of brominated d(C-G)3 are compared to those of the B helix-coil transition observed for methylated d(C-G)3 in 0.1 M NaCl solution while the thermodynamic data of these two reactions (enthalpy and midpoint temperature) are slightly different.     

Conformational and thermodynamic consequences of the introduction of a nick in duplexed DNA fragments: an NMR study augmented by biochemical experiments.

NMR studies were carried out on various equimolar mixtures consisting of a combination of oligomers: d(ACGGCT) (I). d(pACGGCT) (Ia), d(TGCAGT) (II), d(AGCCGTACTGCA) (III), d(TGCAGTACGGCT) (IV). It is shown that I + II + III (MI) and Ia + II + III (M2) form stable duplexes with nicks in the centre of the respective double helices. A close analysis of the NOESY experiments of M1 and M2 revealed that these fragments form B-DNA type duplex structures. A comparison of the chemical-shift data of the nicked duplexes with those of the intact duplex of III + IV (M3) demonstrated that only small local distortions occur when a nick is introduced. The chemical-shift profiles of M1 and M3 were used to obtain the thermodynamic data for the duplex/coil transitions. The profiles of M1 were analysed by means of a new thermodynamic model (TRIDUP). From the calculated thermodynamic data of M1 and M3 it is concluded that the melting behaviour of M1 occurs cooperatively. A ligation experiment demonstrated that the relatively small substrate (M2) was almost completely joined after an overnight incubation at 14 degrees C.     

Investigation of the cAMP receptor protein secondary structure by Raman spectroscopy

Raman spectroscopy was employed to examine the secondary structure of the cAMP receptor protein (CRP). Spectra were obtained over the range 400-1900 cm-1 from solutions of CRP and from CRP-cAMP cocrystals. The spectra of CRP dissolved in 30 mM sodium phosphate and 0.15 M NaCl buffered at either pH 6 or pH 8 or dissolved in 0.15-0.2 M NaCl at protein concentrations of 5, 15, and 30 mg/mL were examined. Estimates of the secondary structure distribution were made by analyzing the amide I region of the spectra (1630-1700 cm-1). CRP secondary structure distributions were essentially the same in either pH and at all protein concentrations examined. The amide I analyses indicated a structural distribution of 44% alpha-helix, 28% beta-strand, 18% turn, and 10% undefined for CRP in solution. Raman spectra of CRP-cAMP cocrystals differed from the spectra of CRP in solution. Some differences were assigned to interfering background bands, whereas other spectral differences were attributed to changes in CRP structure. Differences in the amide III region and in the intensity at 935 cm-1 were consistent with alterations in secondary structure. Analysis of the amide I region of the CRP-cAMP cocrystal spectrum indicated a secondary structure distribution of 37% alpha-helix, 33% beta-strand, 17% turn, and 12% undefined. This result is in agreement with a published secondary structure distribution derived from X-ray analysis of CRP-cAMP cocrystals (37% alpha-helix and 36% beta-strand).(ABSTRACT TRUNCATED AT 250 WORDS)     

Base sequence criteria and Cartesian coordinates for stable B/Z and B/Z/B junctions in relaxed DNA.

It seems increasingly evident that if the Z form of DNA exists in the genome it must exist as short sections of alternating pyrimidine-purine sequences in the midst of very long sections of B-form DNA. We have determined the minimum length of a string of alternating CG base pairs that can go into the Z form in the middle of a long B form. Self-complimentary oligomers of the form T(M)(CG)(N)A(M) were synthesized. The conformation of the resulting duplex was determined in 6M aqueous NaCl solution by Raman scattering. We have found that 12 alternating CG base pairs is the minimum length required to form a stable Z form of DNA inside of a long B form section. Only the 4 center CG base pairs go into the Z form. These 4 CG base pairs in the Z form are flanked on each side by 4 CG base pairs in a non-Z (probably B) form as well as the ..TT.. ..AA.. sequences in the B form. We propose a model of the B/Z junction in which the double helix flips directly from the B form to the Z form so that there are no base pairs in the junction. In this model the B form is nucleated in the AT base pairs on each end and is propagated into the CG base pairs in the center. This model is supported by isotopic H/D exchange experiments that shows that the H/D exchange of the non-Z form CG base pairs is highly retarded and indicates that they remain in the B form. A Thermodynamic analysis of the concentration dependence of the melting point of the duplexes in both low and high salt, supports our model and rules out the possibility of hairpin formation. The enthalpy for the formation of a B/Z junction is determined to be about +16 kcal/junction. A comparison of these results with recent results on B/Z junctions in super-coiled DNA is given. Molecular modeling calculations permit us to obtain values for the coordinates and torsional angles of the oligomers showing both B/Z and B/Z/B junctions. The Cartesian coordinates for these oligomers as well as stereo figures of these models in color are available from the authors.     

Raman spectra of the model B-DNA oligomer d(CGCGAATTCGCG)2 and of the DNA in living salmon sperm show that both have very similar B-type conformations

Raman spectra were obtained from aqueous solutions of the deoxyoligonucleotide d(CGCGAATTCGCG)2 (I), which has been suggested as a model for B-type DNA conformation. These spectra were compared with the Raman spectra of the aqueous solutions of several DNAs of natural origin taken under identical solution conditions. Since the model sequence has a high percent GC (66%), the Raman spectrum was compared with the Raman spectrum of the DNA from Micrococcus lysodeikticus (72% GC), and the spectra of the two different DNAs were found to be rather similar in both 50 mM salt and 6 M salt solutions. Computer-aided band-shape analysis of the backbone vibrational region of the Raman spectra shows the existence of several bands corresponding to different furanose ring puckers. This appears to indicate a heterogeneity of furanose ring pucker in both the model dodecamer and the native DNA. Significant differences were found in the intensity of the conformational marker band at 810 cm-1, which indicates corresponding differences in furanose ring pucker heterogeneities in these two high GC content DNAs. The Raman spectrum of the dodecamer (I) was used to analyze the Raman spectrum of the DNA inside the head of living intact salmon sperm. Sperm spectra were taken with both our conventional Raman spectrograph and a newly developed intracavity laser Raman microscope system. Although the DNA in the sperm head is required by packing considerations to be in a highly compact and condensed state, the Raman spectra of the intact sperm are almost identical with that of the model dodecamer (I) if the difference in base composition is taken into account.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal and solution structures of the B-DNA dodecamer d(CGCAAATTTGCG) probed by Raman spectroscopy: heterogeneity in the crystal structure does not persist in the solution structure

The self-complementary dodecamer d(CGCAAATTTGCG) crystallizes as a double helix of the B form and manifests a Raman spectrum with features not observed in Raman spectra of either DNA solutions or wet DNA fibers. A number of Raman bands are assigned to specific nucleoside sugar and phosphodiester conformations associated with this model B-DNA crystal structure. The Raman bands proposed as markers of the crystalline B-DNA structure are compared and contrasted with previously proposed markers of Z-DNA and A-DNA crystals. The results indicate that the three canonical forms of DNA can be readily distinguished by Raman spectroscopy. However, unlike Z-DNA and A-DNA, which retain their characteristic Raman fingerprints in aqueous solution, the B-DNA Raman spectrum is not completely conserved between crystal and solution states. The Raman spectra reveal greater heterogeneity of nucleoside conformations (sugar puckers) in the DNA molecules of the crystal structure than in those of the solution structure. The results are consistent with conversion of one-third of the dG residues from the C2'-endo/anti conformation in the solution structure to another conformation, deduced to be C1'-exo/anti, in the crystal. The dodecamer crystal also exhibits unusually broad Raman bands at 790 and 820 cm-1, associated with the geometry of the phosphodiester backbone and indicating a wider range of (alpha, zeta) backbone torsion angles in the crystal than in the solution structure. The results suggest that backbone torsion angles in the CGC and GCG sequences, which flank the central AAATTT sequence, are significantly different for crystal and solution structures, the former containing the greater diversity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The concentration dependence of the right to left conformational transition in natural DNA identified by Raman spectroscopy

The classical and resonance Raman spectra of DNA from Chicken Erythrocytes have been obtained for different DNA concentrations in solution with low and high ionic strengths. The classical Raman spectra of 30 mg/ml DNA solutions were measured in varying the sodium chloride concentration from 0.1 to 4.5 M NaCl. An increase in the salt content of the solution leads to spectral changes in the 600-700 cm-1 region, indicating a C2' endo/anti to C3' endo/syn conformational transition of the purine residues. Other changes around 840 cm-1, due to the antisymmetrical stretching vibration of the PO2 group, are also detected: they were characteristic for the B----Z transition in model systems such as poly(dG-dC).poly(dG-dC). The resonance Raman spectra of low (1 mg/ml) and high (30 mg/ml) concentrated DNA solutions were obtained with low (0.1 M) and high (4.5 M) NaCl contents, in using a 284 nm excitation wavelength. No change was observed in the intensities and band positions in the low and high salt solutions of low concentrated DNA. Thus it is assumed that the DNA structure remains unchanged whatever the salt concentration for low concentrated DNA. In contrast, great modifications of the intensities and positions of some lines were found in the spectra of high DNA concentration solution when the NaCl content is increased up to 4.5 M: these changes resemble to some extent those observed in the study of B----Z transition of several polynucleotide model compounds. It is assumed that the right-handed to left-handed conformational transition may occur in certain sections of natural DNA, likely containing alternating purine-pyrimidine sequences, when the DNA concentration is sufficiently important.     

A duplex of the oligonucleotides d(GGGGGTTTTT) and d(AAAAACCCCC) forms an A to B conformational junction in concentrated salt solutions

The coexistence of both A form and B form tracts and formation of an A-B junction in the oligomer d(GGGGGTTTTT).d(AAAAACCCCC) in saturated sodium chloride solution have been detected by Raman spectroscopy. The entire duplex adopts the familiar B-form conformation in aqueous solution at low salt concentrations (0.1M NaCl). In 6M NaCl the adoption of an A form is observed within the G,C tract while a B-form is maintained in the A.T tract. The experimental results indicate that two different helical forms can co-exist in a rather short oligonucleotide and that formation of an A-B junction can occur over a fairly small span of bases. This is in agreement with recent rules governing the relation between base sequence and secondary structure of DNA published from this laboratory. The conformational preferences of each of the individual oligomers d(AAAAACCCCC) and d(GGGGGTTTTT) have also been investigated. The oligomer d(AAAAACCCCC) is single stranded but some evidence for base stacking is observed at 2 degrees C. In contrast, a double stranded B-form structure characterized by wobble G-T base pairing is observed for d(GGGGGTTTTT) in 0.1M and 6M NaCl.     

Anti-Candida activity of four antifungal benzothiazoles

Abstract Anti- Candida activity of 6-amino-2- n -pentylthiobenzothiazole (I), benzylester of (6-amino-2-benzothiazolylthio)acetic acid (II) and of 3-butylthio-(1,2,4-triazolo)-2,3-benzothiazole (III) was followed and compared to that of 2-mercaptobenzothiazole (IV). I and II exhibited good activity against the C. albicans yeast form, similar to IV. They were inhibitorily active against other Candida strains, IC₅₀ values being of the order of 10⁻⁵ M, which means better activity than IV. Compound I also exhibited inhibitory activity on germ-tube formation and mycelial growth in the C. albicans strains, while II, III and IV were not active in these tests. III was the least active form of the compounds tested, IC₅₀ values being of the order of 10⁻⁴ M. All the compounds tested were highly active on a nystatin-resistant C. albicans mutant, with IC₅₀s of the order of 10⁻⁶ M−10⁻⁵ M.